Most of the virulence studies on influenza viruses have focused on the HA, NA and NSl. While these proteins are essential to virulence, they are not the whole story. This proposal will focus on the proteins, mainly PB1-F2 and PB2, which are translated from the polymerase segments, and will investigate their contributions to virulence and transmission. Both of these proteins have been shown to contribute to virulence but their molecular mechanism of virulence and contribution to transmission is unknown. Our laboratory has been at the forefront of studying the function of PB1-F2, a newly discovered virulence factor. We will investigate the contribution of PB1-F2 to the virulence of previous pandemic viruses and the currently circulating H5N1 viruses using the mouse model. In addition, the molecular mechanism of the PBl F2 protein will be examined by analyzing its function as an apoptosis-inducer through protein-protein interaction by immunoprecipitation and stability studies. Finally, we will use the guinea pig model of transmission developed in our laboratory to study the contribution of PB1-F2, PB2 and secondary bacterial infections to transmission. PB1-F2 will be studied using viruses with the expression of PB1-F2 abrogated or with a pathogenicity-enhancing mutation N66S. The PB2 amino acid 627 site, shown to be important of host specificity, will be studied in transmission experiments to find out if and E or K at position 627 impacts transmission. Recently published reports have demonstrated that bacterial pneumonia was a leading cause of death during the 1918 pandemic and may be an important factor for morbidity and mortality in future pandemics. We will therefore conduct transmission experiments with animals that are infected with secondary bacterial pneumonia. Our goal with all of the experiments mentioned above is to elucidate and characterize virulence and transmission factors that have been insufficiently studied in the past. RELEVANCE (See instructions): With the possibility of a 21^' century influenza pandemic, it is crucial to understand the virus as well as we can. The role of the polymerase genes in virulence and transmission has been relatively under-studied, and this project may lead to discoveries which can help to contain a future influenza pandemic.